Last year I constructed a restriction enzyme map of a chimeric plasmid designated pJR4 that was derived from the ligation of the EcoRI "B" fragments from pAM alpha 1 and pAM beta 1. pJR4 is a tetracycline resistance plasmid and is now officially designated as pRAN4. Its molecular size has been revised downward from 8.3 kilobase pairs (kb) to 7.8 kb. This year I examined other putative tetracycline resistance clones for plasmids and found the following. There are to date four classes of plasmids that came out of the original ligated mixture that was introduced by transformation into Streptococcus sanguis, strain Wicky. The first most prevalent class contains plasmids with two EcoRI restriction sites and are composed entirely of the EcoRI B fragments of both pAM alpha 1(3 kb) and pAM beta 1 (5.3kb); an example of this class in pRAN20 (8.3 kb). The second class also contains two EcoRI sites but suffers a deletion in the pAM alpha 1 part of the molecule. An example of this class is pRAN5 (7.9 kb). The third class of plasmids, which also suffers deletions, has single EcoRI sites. Examples of this class are pRAN4 and pRAN1 (7.4 kb). The fourth class of plasmids consists only of the EcoRI B fragment from pAM alpha 1. It also contains a single EcoRI site. Examples are pRAN16, pRAN17, and pRAN24 (all are 3 kb). The first three classes of plasmids are of interest because each contains all or part of separate replicons, namely, those derived from pAM alpha 1 and pAM beta 1. The fourth class contains only the pAM alpha 1 replicon. I am currently trying to ascertain which replicon is the functional one in the first three classes of molecules. I also modified a plasmid purification method that yields native plasmid molecules that are easily cut with a variety of restriction enzymes. The method, in general, deproteinizes total nucleic acids in cell lysates and then denatures chromosomal DNA by use of a buffer at pH 12.3. The method can be used for screening clones for plasmids as well as preparing plasmids in large amounts.